Inspect Demultiplexing

Setup

library(ngsReports)
library(magrittr)
library(scales)
library(pander)
library(tidyverse)
if (interactive()) setwd(here::here("R"))

rawFqc <- list.files("../0_rawData/FastQC/", pattern = "zip", full.names = TRUE) %>%
    FastqcDataList()
trimFqc <- list.files("../1_trimmed/FastQC/", pattern = "zip", full.names = TRUE) %>%
    FastqcDataList()
deMuxFqc <- list.files("../2_demux/FastQC/", pattern = "zip", full.names = TRUE) %>%
    FastqcDataList()
origFqc <- list.files("../previousAnalysis/FastQC/", pattern = "fastqc.zip", full.names = TRUE) %>%
    FastqcDataList()
samples <- read_tsv("../0_rawData/samples.tsv")

Base Qualities

Each of the libraries was inspected for overall quality. Positions 3 & 4 from R2 libraries FCC21WPACXX-CHKPEI13070002_L6 and FCC21WPACXX-CHKPEI13070003_L7 showed clear problems with read qualities. This was likely due to an unsatisfactory nucleotide diversity in the original sequencing run and not enough phiX to overcome this, particularly as all R2 reads will begin with the restriction site (i.e. fragments will terminate with this as there is no R2 barcode).

Base qualities before trimming

Base qualities after trimming

Read Totals

Results from adapter removal. Reads < 70bp after trimming were discarded during this process

Library	Raw	Trimmed	Retained	Discarded
FCC229TACXX-CHKPEI13070001_L3	56,981,567	55,762,854	97.86%	2.14%
FCC2GPDACXX-CHKPEI13070007_L6	66,601,007	64,940,945	97.51%	2.49%
FCC2GPDACXX-CHKPEI13070004_L2	78,411,565	76,201,274	97.18%	2.82%
FCC2GPDACXX-CHKPEI13070006_L4	70,874,412	68,811,191	97.09%	2.91%
FCC21WPACXX-CHKPEI13070003_L7	65,481,942	63,542,676	97.04%	2.96%
FCC2GPDACXX-CHKPEI13070005_L3	78,445,394	76,021,945	96.91%	3.09%
FCC21WPACXX-CHKPEI13070002_L6	69,729,067	67,032,866	96.13%	3.87%
Total	486,524,954	472,313,751	97.1%	2.92%

The first check after demultiplexing is to ensure that read were not assigned to multiple individuals by sabre pe, given that one mismatch was permitted per barcode/restriction-site. Read Totals before and after demultiplexing were then checked and the recovery rate was >93% for each library, with the clear exception of FCC21WPACXX-CHKPEI13070002_L6. Manual inspection revealed that a significant majority (~9.3x10⁶ of 13.4x10⁶) of these non-recovered reads contained truncated barcodes with the first two bases missing. If an additional recovery step was taken this would increase the recovery rate to 93% for this library, and may yield an additional 5-600,000 reads per sample. For the next most poorly recovered library, an additional step may recover an additional 3x10⁶ reads. However, no further action was taken at this point.

trimReadTotals <- readTotals(trimFqc) %>%
    mutate(Library = str_remove_all(Filename, "_[12].fq.gz")) %>%
    distinct(Library, Total_Sequences)
deMuxReadTotals <- readTotals(deMuxFqc) %>%
    mutate(ID = str_remove_all(Filename, ".[12].fq.gz")) %>%
    distinct(ID, Total_Sequences) %>%
    left_join(samples, by = "ID") %>%
    group_by(Library) %>%
    summarise(DeMultiplexed = sum(Total_Sequences)) %>%
    filter(!is.na(Library))

Recovery rate from demultiplexing the 1996 and 2012 samples, after adapter removal

Library	Total Sequences	DeMultiplexed	Recovery Rate
FCC21WPACXX-CHKPEI13070002_L6	67,032,866	53,568,147	79.9%
FCC2GPDACXX-CHKPEI13070005_L3	76,021,945	70,702,537	93.0%
FCC2GPDACXX-CHKPEI13070006_L4	68,811,191	66,677,690	96.9%
FCC229TACXX-CHKPEI13070001_L3	55,762,854	55,037,971	98.7%
FCC21WPACXX-CHKPEI13070003_L7	63,542,676	62,787,380	98.8%
FCC2GPDACXX-CHKPEI13070004_L2	76,201,274	75,316,445	98.8%
FCC2GPDACXX-CHKPEI13070007_L6	64,940,945	64,349,296	99.1%
Total	472,313,751	448,439,466	94.9%

Read Totals for each of the 1996 & 2012 samples. The black line indicates the mean library size, whilst the dashed green line indicates the mean library size based on the original library before demultiplexing. Bar colours indicate sample population as 1996 (blue) or 2012 (red).

In summary, of the 486,524,954 initial reads, a total of 448,439,466 were retained after trimming and demultiplexing. This represents a combined recovery rate of 92.2% from the initial libraries.

Comparison against previous analysis

The revised strategy showed considerable improvement in yield for the libraries FCC21WPACXX-CHKPEI13070002_L6 and FCC21WPACXX-CHKPEI13070003_L7 which both contained exculsively 1996 samples. As such the revised strategy was pursued further.

Comparison of library sizes after demultiplexing using sabre (blue), vs the original method (salmon).

Changes in library size using the improved recovery strategy

Comparison of library sizes for the three populations.

GC Content

GC content for all 1996 and 2012 samples.

Inspection by GC content showed that gc2709 and gc2700 appeared to have an exaggerated peak around 60%, whilst all other samples showed a more broad spread across the range. From the Turretfield samples (previous analysis), pt1125 showed an unexpected peak around 50% indicating that sample may contain reads from a different species. This sample should be excluded from all further analysis.

GC content for all Turretfield samples.

Read Lengths

Length Distribution for all files

Sequence Content

Sequence content showing the presence of the RS in both the Turretfield and R2 samples.

Conclusion

• The restriction site should be removed from both R2 and Turretfield samples, especially given the high error rate at positions 2/3 in some R2 samples
• No further processing is required as the presence of the restriction site at both ends means fixed length reads are not required for Stacks